Cell Functional State Research Articles

HLA-DR Expression in Natural Killer Cells Marks Distinct Functional States, Depending on Cell Differentiation Stage.

HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.

Single-Cell Mechanics: Structural Determinants and Functional Relevance.

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications.

Erythroid Cells as Full Participants in the Tumor Microenvironment.

The tumor microenvironment is an important factor that can determine the success or failure of antitumor therapy. Cells of hematopoietic origin are one of the most important mediators of the tumor-host interaction and, depending on the cell type and functional state, exert pro- or antitumor effects in the tumor microenvironment or in adjacent tissues. Erythroid cells can be full members of the tumor microenvironment and exhibit immunoregulatory properties. Tumor growth is accompanied by the need to obtain growth factors and oxygen, which stimulates the appearance of the foci of extramedullary erythropoiesis. Tumor cells create conditions to maintain the long-term proliferation and viability of erythroid cells. In turn, tumor erythroid cells have a number of mechanisms to suppress the antitumor immune response. This review considers current data on the existence of erythroid cells in the tumor microenvironment, formation of angiogenic clusters, and creation of optimal conditions for tumor growth. Despite being the most important life-support function of the body, erythroid cells support tumor growth and do not work against it. The study of various signaling mechanisms linking tumor growth with the mobilization of erythroid cells and the phenotypic and functional differences between erythroid cells of different origin allows us to identify potential targets for immunotherapy.

SABRSeq: A Randomized Phase Ib Trial of SABR Sequencing with Pembrolizumab in Metastatic Non-Small Cell Lung Cancer (NSCLC)

The optimal sequencing of stereotactic ablative body radiotherapy (SABR) with anti-PD1 checkpoint blockade is unknown. The purpose of SABRseq was to assess the toxicity of the combination of SABR delivered either before or after the commencement of pembrolizumab. The central hypothesis is that the treatment combination will have a safety profile that is clinically acceptable and demonstrate anti-tumor efficacy. Furthermore, we hypothesize that differences in systemic immune activation will be observed between treatment arms. This was a single institutional phase Ib randomized clinical trial (Trial ID NCT03307759). Eligible patients had metastatic NSCLC, ECOG performance 0-1, and had not received prior checkpoint immunotherapy, high-dose radiation (>36 Gy) within 6 months, or either systemic or radiation therapy within 4 weeks of randomization. Eligible patients had either TPS≥50% PD-L1 expression in the first-line or TPS≥1% PD-L1 expression with prior chemotherapy exposure. Patients were randomized to SABR before the commencement of pembrolizumab [ARM1] or SABR commencing after pembrolizumab [ARM2]. SABR was delivered in a single fraction of 18-20 Gy to 1-3 lesions. The primary endpoint was treatment-related adverse events (AE's) related to SABR and/or pembrolizumab. Secondary endpoints included best overall response (BOR), overall survival (OS), and progression-free survival (PFS). Translational objectives included the evaluation of longitudinal changes in immunological cellular subsets within peripheral blood to explore changes in systemic immunity and circulating tumor DNA (ctDNA) dynamics. Between December 2017 and December 2019, 13 patients were randomized. The median follow-up was 37 months. The study was closed early due to poor accrual. Median age was 66 years, with 11 patients (84%) having adenocarcinoma. Nine (69%) were enrolled in the first-line setting. The median [range] number of lesions was 6 [3-11]. The median [range] cycles of pembrolizumab delivered in ARM 1 was 13 [12-32] and ARM 2 was 9 [3-34]. Grade 3 treatment-related AEs were experienced in 0 of 5 patients in ARM1 and in 1 of 8 patients in ARM 2 (hyperglycemia). There were no grade 4 or 5 adverse events reported. The BOR by both RECIST and iRECIST criteria was CR in one patient, PR in seven patients, SD in five patients. Median (95% Cis) PFS was 12.4 months (6.3-21.0), and median (95% Cis) OS was 47.1 months (12.6-not reached; 2-year point estimate 62% [31-82]). Mass cytometry was used on serial peripheral blood samples to examine changes in the frequency of immune cells, changes in T cell activation, differentiation and functional polarization state. Targeted sequencing was performed to assess ctDNA. Translational outcomes will be presented. There was no evidence of a concerning safety signal from either SABR before or after start of pembrolizumab. The combination demonstrated activity with promising PFS and OS and is worthy of evaluation in larger randomized trials.

Physical Training Chronically Stimulates the Motor Neuron Cell Nucleus in the Ts65Dn Mouse, a Model of Down Syndrome.

Down syndrome (DS) is a genetically-based disease based on the trisomy of chromosome 21 (Hsa21). DS is characterized by intellectual disability in association with several pathological traits among which early aging and altered motor coordination are prominent. Physical training or passive exercise were found to be useful in counteracting motor impairment in DS subjects. In this study we used the Ts65Dn mouse, a widely accepted animal model of DS, to investigate the ultrastructural architecture of the medullary motor neuron cell nucleus taken as marker of the cell functional state. Using transmission electron microscopy, ultrastructural morphometry, and immunocytochemistry we carried out a detailed investigation of possible trisomy-related alteration(s) of nuclear constituents, which are known to vary their amount and distribution as a function of nuclear activity, as well as the effect of adapted physical training upon them. Results demonstrated that trisomy per se affects nuclear constituents to a limited extent; however, adapted physical training is able to chronically stimulate pre-mRNA transcription and processing activity in motor neuron nuclei of trisomic mice, although to a lesser extent than in their euploid mates. These findings are a step towards understanding the mechanisms underlying the positive effect of physical activity in DS.

Abstract 3276: Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the US partly because of its resistance to conventional chemotherapies. Immunotherapies that improve survival in many cancers, almost universally fail in PDA. The resistance to immune checkpoint blockade is at least partially explained by the lack of T cells in the tumor microenvironment (TME) in PDA. We have previously demonstrated that tumor cell-derived prostaglandin E2 (PGE2) is one of the tumor intrinsic factors that creates/supports immunosuppressive TME in PDA presumably through its paracrine effect on the stromal cells. In the current study, we investigate the role of autocrine PGE2 signaling through its cognate EP4 receptor in shaping the T cell low, myeloid cell rich PDA TME. The knockdown (EP4 KD) or knockout (EP4 KO) of the receptors on KrasG12D/p53R172H/YFP/PdxCre (KPCY) tumor cells was sufficient to suppress or abolish the growth of implanted tumors, respectively (control vs EP4 KD, p=0.01, and control vs EP4 KO, p<0.0001). The rejection of the EP4 KO PDA cell lines was T cell dependent as it did not happen in hosts depleted of CD4+ and CD8+ T cells. Upon rechallenge, 80-90% of mice that rejected the primary EP4 KO tumors were able to reject the control, EP4-sufficient tumors in a T cell-dependent manner. While EP4 KO tumors were universally rejected by wild-type (WT) or EP4 global KO (KOgl) hosts, control cell lines formed tumors and grew in both hosts albeit with significantly slower growth in EP4 KOgl hosts (p<0.0001). The TME analysis revealed changes in the T cell presence, subtypes, and functional state in EP4 KO tumors demonstrating more T cell inflamed TME with increased proportions of central memory CD4+ and CD8+ cells (p=0.02 both) along with downregulation of exhaustion markers PD-1 (p=0.02) and CD39 (p=0.009) on CD4+ and CD8+ cells, respectively. Deletion of EP4 on tumor cells changed the systemic anti-tumor immunity by inducing T cell proliferation in lymph nodes (EP4 KO vs control, Ki67+CD8+ p<0.0001 and Ki67+CD4+ p=0.003) and depleting gMDSC population in the spleens of EP4 KO tumor bearing mice (p<0.0001). RNAseq analysis of tumor cells (cultured and in vivo) as well as bulk tumors identified TNFa, IFNa, and IFNg pathways as top upregulated ones in EP4 KO tumors while TGFb and Myc pathways were among the significantly downregulated pathways. The study demonstrates a primary role of tumor cell PGE2-EP4 signaling in creating immunosuppressive TME in PDA suggesting EP4 blockade as a novel approach in treatment of pancreatic cancer. Citation Format: Nune Markosyan, Charu Arora, Liz Quinones, Il-Kyu Kim, Nikhil Joshi, Noah Cheng, John Tobias, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3276.

Regulon active landscape reveals cell development and functional state changes of human primary osteoblasts in vivo

BackgroundWhile transcription factor (TF) regulation is known to play an important role in osteoblast development, differentiation, and bone metabolism, the molecular features of TFs in human osteoblasts at the single-cell resolution level have not yet been characterized. Here, we identified modules (regulons) of co-regulated genes by applying single-cell regulatory network inference and clustering to the single-cell RNA sequencing profiles of human osteoblasts. We also performed cell-specific network (CSN) analysis, reconstructed regulon activity-based osteoblast development trajectories, and validated the functions of important regulons both in vivo and in vitro.ResultsWe identified four cell clusters: preosteoblast-S1, preosteoblast-S2, intermediate osteoblasts, and mature osteoblasts. CSN analysis results and regulon activity-based osteoblast development trajectories revealed cell development and functional state changes of osteoblasts. CREM and FOSL2 regulons were mainly active in preosteoblast-S1, FOXC2 regulons were mainly active in intermediate osteoblast, and RUNX2 and CREB3L1 regulons were most active in mature osteoblasts.ConclusionsThis is the first study to describe the unique features of human osteoblasts in vivo based on cellular regulon active landscapes. Functional state changes of CREM, FOSL2, FOXC2, RUNX2, and CREB3L1 regulons regarding immunity, cell proliferation, and differentiation identified the important cell stages or subtypes that may be predominantly affected by bone metabolism disorders. These findings may lead to a deeper understanding of the mechanisms underlying bone metabolism and associated diseases.

Blockades of effector T cell senescence and exhaustion synergistically enhance antitumor immunity and immunotherapy

BackgroundCurrent immunotherapies still have limited successful rates among cancers. It is now recognized that T cell functional state in the tumor microenvironment (TME) is a key determinant for effective antitumor...

Abstract 1235: Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor - T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced non-small cell lung cancer (NSCLC). Although, other immune cell subsets were shown to be associated with clinical efficacy, their relative impact and combined effect in predicting improved long-term survival warrant further investigation. Using computational image analysis of multiplex immunofluorescence (mIF) and immunohistochemistry (IHC) immune marker panels, we sought to identify single and combined biomarkers of the tumor immune contexture in association with long-term OS in advanced NSCLC patients treated with Durvalumab. Methods: Pre-treatment tumor samples from advanced NSCLC patients (n = 210) enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562), were stained using IHC and 6-marker mIF panels to detect markers of immune cells, cell functional state and tertiary lymphoid structure (TLS) (PD-L1, CD8, PD-1, Ki67, CD68, CD20, CD1c, NKp46, CD66b, ICOS, FOXP3). Cell marker density (cells/mm2), distribution and proximity were quantified and analyzed in association with overall survival. Results: Computational image analysis of the tumor immune contexture revealed a greater immune inflamed phenotype, both innate (macrophages, dendritic cells) and adaptive (T and B cells), in NSCLC patients with long OS >2 years compared to those with short OS < 1 year (fold change > 2, p < 0.0001). Patient subgroup with high density of individual immune subsets show a median OS (mOS) of 10-20 months (p < 0.01 high vs. low subgroups) while combined markers of innate and adaptive immune cells show an improved mOS > 2 years (p < 0.001). Specifically, among the key findings, combined biomarkers of CD68+ PD-L1+ macrophages and CD8+ T cells predicts for a significant increase in OS for patients with high vs low marker density (HR = 0.21, 95%CI 0.12 - 0.39, p <10-7; mOS 39.5 months [high], 6.5 months [low]). Whereas, in patients with high density of either single biomarkers CD68+ PD-L1+ macrophage or CD8+ T cells mOS is 20.2 or 18.4 months respectively. Moreover, high density of CD20+ B cells, reflective of presence of TLS, associates with improved OS (mOS NR [high], 11 months [low], p = 0.003). In addition, TLS enriched tumors show an increased level of macrophages expressing PD-L1 in synapsis with CD8+ T cells (activated PD1+ or proliferative Ki67+ T cells). Conclusion: These findings demonstrate the importance of both tertiary lymphoid structure and high pre-existing innate-adaptive immunity in driving long-term overall survival of durvalumab-treated patients with NSCLC and highlight the need for the development of multiparametric predictive biomarkers beyond tumor-T cell axis. Citation Format: Lina Meinecke, Jorge Blando, Thomas Herz, Michael Surace, Thomas Padel, Monica Azqueta Gavaldon, Harald Hessel, Farzad Sekhavati, Megha Saraiya, Anmarie Boutrin, Karma Da Costa, Jaime Rodriguez Canales, Ashok Gupta, Carl Barrett, Zachary Aaron Cooper, Ikbel Achour. Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1235.

Comparative characteristics of DNA loop domains rearrangement in glioblastoma multiforme T98G and glioblastoma astrocytoma U373 cell lines underdifferent culture conditions.

The loop domain organization of chromatin, which plays an important role in transcription regulation, may depend on the cell functional state. The aim of this work was to investigate DNA loop reorganization upon functional transitions in two cell lines‒ glioblastoma multiforme T98G and glioblastoma astrocytoma U373. Single cell gel electrophoresis (the comet assay) was used to analyze the kinetics of the DNA loop migration from the nucleoids obtained from the lysed cells. The DNA fraction in the surface loops and the size of these loops were found to be similar in two glioblastoma cell lines. When synthetic processes were inhibited, the migration of a small portion of inner loops was observed in T98G but not U373cells. In T98G cells, stimulation of cell proliferation and transcription was accompanied by an increase in DNA fraction in the inner loops and an essential increase in the size of these loops. The effect of stimulation was practically absent in U373cells. However, the linear density of the loops resolved by the comet assay was found to decrease upon stimulation of proliferation in both cell lines. A decrease in the loop density appears to be associated with an intensification of the synthetic processes in cells upon their stimulation.

Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)

BackgroundMetabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization.ResultsWe aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions.ConclusionThe validation of the method consisting in a JURKAT cell line experimental incorporation as a control material contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

Transcriptomic and Epigenomic Regulation of Leukocytes by the Polyamine Pathway

Abstract Cellular metabolism can orchestrate immune cell function. We previously demonstrated that lipid biosynthesis represents one such gatekeeper to Th17 cell functional state, promoting autoimmune responses in the experimental autoimmune encephalomyelitis model. Utilizing a transcriptome-based insilico fluxomics tool, we investigated ancillary metabolic pathways associated with Th17 cell function and studied in detail the relevance of the polyamine pathway as modulators of autoimmune inflammation. We investigated this pathway using carbon-tracing fluxomics, transcriptomics, epigenomics (ATACseq) and functionally validated their relevance in autoimmune disease models using small molecule inhibitors and knockout animals.

The Behavior of Monolayer Zajdela Hepatoma Cells in an Experimental Wound Space

Long-term cultivated mammalian cell lines, as a rule, have either an epithelial- or fibroblast-like morphology. The monolayer cell line that we obtained from rat ascites Zajdela hepatoma contains cells of both types, genetically related but phenotypically different. We compared the behaviors of these cells during their migration into the free space of an experimental “wound” under the same cultivation conditions. We measured the following parameters: the dynamics of the cell number in this space, area of cell projection on the substrate, coefficient of cell spreading, rate of cell migration, and proposed parameter W reflecting the cell efficacy in the wound closure. We found that, initially (at the first stage, within 9–10 h), wound closure was due to the cell migration and increased area of cell projection on the substrate. At the second stage, the cell division contributed to a further increase in cell number within the wound. In terms of parameter W, epithelial-like cells moved less chaotically and filled the wound approximately 1.5 times more efficiently than fibroblast-like cells, the motion of which was alike to Brownian. Meanwhile, the average migration rate of epithelial-like cells was about half that of fibroblast-like cells. In the area of the wound that had just closed, the sizes of cells were larger than those of their counterparts outside the wound. We suggest that the increase in cell size was transitory and resulted from changes in the cell functional state during migration to the wound.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques.

Abstract 2104: In vitro drug effects on cancer cell morphology and functional state revealed by multiparameter imaging mass cytometry

Abstract Results of in vitro drug testing are correlated with clinical response to chemotherapy: Accuracy to predict clinical drug resistance was found to be as high as 90%. The benefit of using in vitro models lies in the ability to probe cellular response in a controlled closed system, where effects of drug concentrations, treatment duration, drug efflux kinetics and multidrug combinations can be assessed by a variety of cell biology techniques. Cisplatin is a widely used chemotherapy drug that targets genomic DNA of the cells, forming both interstrand and intrastrand cross-links that lead to cell death. One limitation of its clinical use is in predicting the development of resistance and severe side effects in patients. Mechanisms of resistance such as reduced drug accumulation, increased detoxification through cisplatin binding to cellular thiols, reduced DNA platination, and increased DNA repair have been reported, however (tamoxifen enhancement of cisplatin). In vitro models of different cancer types (SKOV3, HeLa, A431, MCF-7) were used to study the effects of cisplatin on cell morphology and phenotypic and functional characteristics with a large panel of metal-tagged antibodies and Imaging Mass Cytometry (IMC) at the single-cell level (1). Proteins involved in DNA damage repair (γH2AX, PP2A, pHistone H3), apoptosis (CD98, caspase-3, cleaved PARP), cell proliferation (cyclin B1, Ki-67), metastasis (vimentin, β-catenin, VEGF, CD63, CD9), substrate adhesion (CD29, CD49e, CD49b, CD51, CD54, CD47, CD61), organelle morphology (CD107a, Mito, histone H3), and signaling pathways (STAT3, pERK1/2, pS6), as well as surface receptors (EGFR, HER2, BRCA, MUC1, CD44, EpCAM, CD142, CD59, beta-catenin) and structural markers (CK5, CK8/18, β-actin, β-tubulin), were identified simultaneously in each individual cell with specific metal-conjugated antibodies. S-phase cells were visualized by detection of 127I in 5-iodo-2′-deoxyuridine (IdU) added to culture media. Presence of cisplatin in cell nuclei and cytoplasm was registered by IMC of platinum stable isotopes. Combination therapy of cisplatin and paclitaxel is a standard chemotherapeutic regimen to treat recurrent or metastatic cervical cancer. Cell lines from various tumors may develop resistance to cisplatin. Reduced cisplatin uptake has been observed in cervical cancer cells with cisplatin resistance. The cisplatin-resistant HeLa cells and A431 (A431/Pt) cells show 50% and 77% reduction in cisplatin uptake, respectively, compared with the parental cell lines. Human ovarian cancers (in vitro model SKOV3), for which cisplatin is a mainstay of treatment, develop drug resistance and pose an important clinical challenge. (1) Chang, Q et al. Nature Scientific Reports 6 (2016): 36641 Citation Format: Olga Ornatsky, Alexandre Bouzekri, Bedilu Allo, Jessica Watson. In vitro drug effects on cancer cell morphology and functional state revealed by multiparameter imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2017-2104

Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2.

At higher order levels chromatin fibers in interphase nuclei are organized into loop domains. Gene regulatory elements (promoters and enhancers) are often located near the sites of loop attachments. Therefore, loop domains play a key role in regulation of cell transcriptional activity. We investigated the kinetics of DNA loop exit during single cell gel electrophoresis (the comet assay) of nucleoids obtained from two cell types that differ in their synthetic activity – human lymphocytes and lymphoblasts. Lymphocyte activation and transformation into lymphoblasts (blast transformation) was performed with interleukin 2. The results obtained suggest that a rearrangement of the loops occurs after lymphocyte activation. After blast transformation we observed an increase of the amount of loop domains on the surface of nucleoids against a decrease of the inner loop fraction. Therefore, the comet assay can be used for detection of large-scale changes in the cell nucleus that follow changes in cell functional state.

CD5L/AIM Regulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity

Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis but can induce autoimmunity. The mechanisms implicated in balancing "pathogenic" and "non-pathogenic" Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in non-pathogenic, but not in pathogenic Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts non-pathogenic Th17 cells into pathogenic cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition and restricting cholesterol biosynthesis and, thus, ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.

Toward high-content screening of mitochondrial morphology and membrane potential in living cells

Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These “mitochondrial dynamics” are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial “health” and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Effect of sunlight transformed by luminophore-containing materials on cell functions in vitro and in vivo.

Effect of sunlight transformed by luminophore-containing materials on cell viability and functional state of the retina was assessed using the photodamage model. Exposure to the luminescent component of light improved viability of NIH 3T3 cells and promoted recovery of electric activity in rabbit retina after photodamage.

A model for the generation and interconversion of ER morphologies.

The peripheral endoplasmic reticulum (ER) forms different morphologies composed of tubules and sheets. Proteins such as the reticulons shape the ER by stabilizing the high membrane curvature in cross-sections of tubules and sheet edges. Here, we show that membrane curvature along the edge lines is also critical for ER shaping. We describe a theoretical model that explains virtually all observed ER morphologies. The model is based on two types of curvature-stabilizing proteins that generate either straight or negatively curved edge lines (R- and S-type proteins). Dependent on the concentrations of R- and S-type proteins, membrane morphologies can be generated that consist of tubules, sheets, sheet fenestrations, and sheet stacks with helicoidal connections. We propose that reticulons 4a/b are representatives of R-type proteins that favor tubules and outer edges of sheets. Lunapark is an example of S-type proteins that promote junctions between tubules and sheets. In a tubular ER network, lunapark stabilizes three-way junctions, i.e., small triangular sheets with concave edges. The model agrees with experimental observations and explains how curvature-stabilizing proteins determine ER morphology.